Resolvin E1 promotes phagocytosis-induced neutrophilapoptosis and accelerates resolution ofpulmonary inflammationDriss El Kebira, Per Gjorstrupb, and János G. Filepa,1

aResearch Center, Maisonneuve-Rosemont Hospital, and Department of Pathology and Cell Biology, University of Montréal, Montréal, QC, Canada H1T 2M4;and bResolvyx Pharmaceuticals, Cambridge, MA 02142

Edited by* Charles N. Serhan, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, and approved August 6, 2012 (received for reviewApril 19, 2012)

Inappropriate neutrophil activation contributes to the pathogen-esis of acute lung injury (ALI). Apoptosis is essential for removal ofneutrophils from inflamed tissues and timely resolution of in-flammation. Resolvin E1 (RvE1) is an endogenous lipid mediatorderived from the ω-3 polyunsaturated fatty acid eicosapentaenoicacid that displays proresolving actions. Because the balance ofprosurvival and proapoptosis signals determines the fate of neu-trophils, we investigated the impact of RvE1 on neutrophil apopto-sis and the outcome of neutrophil-mediated pulmonary inflamma-tion in mice. Culture of human neutrophils with RvE1 acceleratedapoptosis evoked by phagocytosis of opsonized Escherichia colior yeast. RvE1 through the leukotriene B4 receptor BLT1 enhancedNADPH oxidase-derived reactive oxygen species generation andsubsequent activation of caspase-8 and caspase-3. RvE1 also atten-uated ERK and Akt-mediated apoptosis-suppressing signals frommyeloperoxidase, serum amyloid A, and bacterial DNA, shiftingthe balance of pro- and anti-survival signals toward apoptosisvia induction of mitochondrial dysfunction. In mice, RvE1 treatmentenhanced the resolution of established neutrophil-mediated pulmo-nary injury evoked by intratracheal instillation or i.p. administrationof live E. coli or intratracheal instillation of carrageenan plus mye-loperoxidase via facilitating neutrophil apoptosis and their removalby macrophages. The actions of RvE1 were prevented by the pan-caspase inhibitor zVAD-fmk. These results identify a mechanism,promotion of phagocytosis-induced neutrophil apoptosis and mit-igation of potent anti-apoptosis signals, by which RvE1 could en-hance resolution of acute lung inflammation.

leukocytes | efferocytosis | innate immunity | resolution of inflammation

Acute inflammation is a tightly regulated self-limited protectiveresponse against invading pathogens and tissue injury. Neu-

trophils, recruited from the circulation, play a prominent role inhost defense (1). Neutralization of the offending insult ideallyprompts resolution of inflammation and restoration of tissue ho-meostasis. Effective resolution of inflammation critically dependson inhibition of neutrophil influx and timely removal of infiltratingneutrophils that is governed by active resolution programs (2).Failure of the initial acute inflammatory response to resolve ina timely manner is now considered as a characteristic feature ofmany common diseases, including acute lung injury or the acuterespiratory distress syndrome (3).Neutrophil apoptosis has emerged as a critical control point in

resolving inflammation. Emigrated neutrophils undergo apoptosisbefore being removedby scavengermacrophages (4, 5). The fate ofneutrophils can, however, be profoundly influenced by prosurvivaland proapoptosis cues from the inflammatory microenvironment.Recent studies using a variety of gene knockout, transgenic, andpharmacological strategies in diverse models of inflammation,including acute lung injury (ALI) and sepsis showed that modu-lating neutrophil apoptosis can profoundly affect the outcome ofinflammation. For instance, delaying neutrophil apoptosis bymyeloperoxidase (MPO) prolonged lung injury (6), whereascyclin-dependent kinase inhibitor drugs (7, 8) or aspirin-triggered

15-epi-LXA4 (9) augmented neutrophil apoptosis parallel withenhancing resolution. Suppressed neutrophil apoptosis appears tobe a component of the pathophysiology in patients with acute re-spiratory distress syndrome (10) and sepsis (11).Resolvin E1 (RvE1) is biosynthesized from the ω-3 poly-

unsaturated fatty acid eicosapentaenoic acid (EPA) during theresolution phase of acute inflammation with leukocyte 5-lip-oxygenase playing a pivotal temporal role in the biosynthesispathway (12, 13). RvE1 binds to ChemR23 and the leukotriene B4(LTB4) receptorBLT1(14,15) andattenuatesneutrophilmigration(2), reduces production of inflammatory cytokines, enhances gen-eration of the proresolving mediator lipoxin A4 (16) and phagocy-tosis of apoptotic neutrophils by macrophages (13), therebyattenuating inflammation in several disease models, includingperitonitis (13), polymicrobial sepsis (17), bacterial pneumonia(18), and allergic airway inflammation (19, 20). Little is knownabout whether RvE1 could affect neutrophil apoptosis, a criticalcontrol point in resolution of inflammation. Here, we report thatRvE1 shortened the life span of human neutrophils by acceleratingphagocytosis-induced neutrophil death and by mitigating the anti-apoptosis signal from diverse inflammatory mediators. Moreover,RvE1 administered at the peak of inflammation augmented cas-pase-mediated neutrophil apoptosis and enhanced inflammationresolution in three murine models of acute lung injury.

ResultsRvE1 Enhances Phagocytosis-InducedNeutrophil Apoptosis andMitigatesProsurvival Signals. Confirming previous observations (17), RvE1 atlower concentrations (1–50 nM) did not affect apoptosis of naiveisolated neutrophils. However, at higher pharmacological concen-trations (100–1,000 nM), it prolonged neutrophil longevity by de-laying intrinsic apoptosis (Fig. 1A). This was associated with modestelevations in reactive oxygen species (ROS) generation (Fig. 1Band Fig. S1) and caspase-8 activity (Fig. 1C) and robust increases inphosphorylation of ERK (Fig. S1). In subsequent experiments, weused RvE1 at concentrations ≤10 nM.Earlier studies showed that RvE1 enhances phagocytosis of

bacteria and apoptotic cells by macrophages (13, 14, 17). We usedopsonized E. coli and yeast as target pathogens to evaluate theimpact of RvE1 on phagocytosis-induced apoptosis. RvE1-en-hanced phagocytosis of FITC-labeled opsonized E. coli by humanneutrophils (Fig. 2 A and B). Consistent with published data (21),neutrophils cultured for 24 h with opsonized yeast underwentapoptosis (Fig. 2C). Phagocytosis evoked a rapid, robust ROSproduction and increased caspase-8 and caspase-3 activity (Fig. 2

Author contributions: D.E.K. and J.G.F. designed research; D.E.K. and J.G.F. performedresearch; P.G. contributed new reagents/analytic tools; D.E.K. and J.G.F. analyzed data;and D.E.K., P.G., and J.G.F. wrote the paper.

Conflict of interest statement: P.G. is president of Resolvyx Pharmaceuticals and retainsfounder stock in the company.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: janos.g.filep@umontreal.ca.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1206641109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1206641109 PNAS | September 11, 2012 | vol. 109 | no. 37 | 14983–14988

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D–F). These were further enhanced by RvE1, resulting in aug-mented neutrophil apoptosis (Fig. 2C). RvE1 augmentation ofapoptosis was even more pronounced at 4-h culture (Fig. S2). TheRvE1 actions were prevented by the NADPH oxidase inhibitordiphenyleneiodonium (DPI) or the BLT1 antagonist U75302, butnot the BLT2 antagonists LY255238 (Fig. 2C).We confirmed thatneutrophils express high levels of BLT1, whereas ChemR23 ex-pression was undetectable (Fig. S3).Because RvE1 induces ROS production and ROS may con-

tribute to constitutive neutrophil apoptosis (22), we investigatedwhether RvE1 could interfere with survival signals from theneutrophil granule enzyme MPO, the acute-phase protein serumamyloid A (SAA) or the bacterial constituent CpG DNA. Wehave chosen these mediators because of their relevance to acutelung injury (23–25) and their known neutrophil apoptosis-sup-pressing action through the β2-integrin Mac-1 (6), formyl-peptidereceptor 2 (FPR2)/lipoxin A receptor (ALX) (26), and toll-likereceptor 9 (TLR9) (27), respectively. As anticipated, MPO (Fig.3A), SAA, and CpG DNA (Fig. S4) prevented disruption ofmitochondrial transmembrane potential (ΔΨm), which precedesdevelopment of apoptotic morphology in neutrophils undergoingconstitutive apoptosis (26, 28). Exposure of neutrophils to RvE1(1–10 nM) mitigated the intracellular survival signals producedfrom MPO (Fig. 3B), SAA, or CpG DNA (Fig. S4), and reducedneutrophil survival by promoting apoptosis. Because MPO, SAA,and CpG DNA evoke rapid phosphorylation of ERK 1/2 andAkt, leading to preservation of Mcl-1 (6, 26, 27), a key regulatorof neutrophil apoptosis (29), we further probed the effects ofRvE1 on these signaling pathways. RvE1 exerted modest in-hibitory actions on ERK and Akt phosphorylation, and Mcl-1expression evoked by MPO (Fig. 3B), SAA, or CpG DNA (Fig.S4). No significant changes were detected in p38 MAPK phos-phorylation (Fig. S4). Furthermore, RvE1 attenuated MPO-

stimulated Mac-1 expression on neutrophils in a concentration-dependent fashion (Fig. S5). MPO, SAA, and CpG DNA at-tenuated activation of caspase-8 that occurs during spontaneousneutrophil apoptosis, and this was almost completely preventedby RvE1 (Fig. S6). Caspase-8 activation is attributed to NADPHoxidase-derived ROS (21, 30). RvE1 reversal of survival signalsfrom MPO, SAA, and CpG DNA was blocked with apocynin andDPI (Fig. S6), suggesting that RvE1 also activated this pathway.RvE1 did not affect Fas-induced neutrophil apoptosis (Fig. S7).

RvE1 Promotes Neutrophil Apoptosis and Enhances Resolution of LungInflammation. Having shown the ability of RvE1 to promoteneutrophil apoptosis in vitro, we investigated the impact of RvE1treatment on the resolution of inflammation in three mousemodels of neutrophil-mediated acute lung injury. In the E. colipneumonia model, treatment with RvE1 reduced neutrophilaccumulation in the airways (Fig. 4 A and B) and increased bron-choalveolar lavage (BAL) fluid monocyte/macrophage numbers(Fig. 4C) concomitant with increases in the number of apoptoticneutrophils (Fig. 4 D–F) and macrophages containing apoptoticbodies (Fig. 4G). Antiinflammatory actions of RvE1 were alsoevident, as indicated by markedly reduced inflammatory cell in-filtrate (Fig. 5A), attenuation of edema formation (Fig. 5 B andC), and BAL fluid IL-6 level (Fig. 5D) and less-severe lung injury(Fig. 5E). The i.p. administration of the pan-caspase inhibitorzVAD-fmk prevented the beneficial actions of RvE1 (Figs. 4 and5), indicating a critical role of neutrophil apoptosis in these events.In the E. coli peritonitis–associated ALI model, treatment with

RvE1 decreased BAL fluid neutrophil numbers without affectingmonocyte/macrophage numbers (Fig. 6A and Fig. S8) and re-duced lung MPO content (Fig. S8) with concomitant increases inthe number of apoptotic neutrophils (Fig. 6 B and C). RvE1 alsoincreased the number of macrophages containing apoptoticbodies (Fig. 6D), attenuated pulmonary edema and BAL fluidIL-6 (Fig. S8), and tissue injury (Fig. 6E). E. coli (109) challengeresulted in a mortality rate of 70% within 6 h; whereas, 70% ofRvE1-treated mice were alive at 6 h (Fig. 6F).The proresolving action of RvE1 was also investigated in the

carrageenan plus MPO instillation-induced lung injury model. Inthis model, carrageenan evokes a spontaneously self-resolvinginflammation that can be prolonged by coadministration of MPOparallel with suppression of neutrophil apoptosis (6). Treatmentwith RvE1 facilitated resolution of established inflammationwhen administered at near the peak of inflammation (24 h) (Fig.S9). RvE1 decreased BAL fluid total leukocyte and neutrophilnumbers with increases in the number of monocytes/macro-phages (Fig. S9). Reduced neutrophil accumulation occurredparallel with increases in the number of apoptotic neutrophils asassessed by positive staining for annexin V, increased caspase-3activity, the amount of cytoplasmic histone-associated DNAfragments, and collapse of ΔΨm (Fig. S9). RvE1 increased thenumber of macrophages containing apoptotic bodies, reducededema formation, BAL fluid IL-6 level, and lung injury (Fig. S9).Coadministration of zVAD-fmk with RvE1 rendered animalsresistant to treatment with RvE1 (Fig. S9).

DiscussionClearance of neutrophils from inflamed tissues is fundamental toresolution of inflammation. Omega-3 polyunsaturated fatty acid–derived lipid mediators, including RvE1, formed during the reso-lution phase of acute inflammation attenuate proinflammatorymediator responses (13, 16–18) and neutrophil infiltration, andenhance phagocytosis of apoptotic cells (2, 31). The present re-sults indicate that by promoting apoptosis in neutrophils in situ,RvE1 enhances resolution of inflammation in three different mousemodels of ALI/acute respiratory distress syndrome (ARDS). RvE1augments phagocytosis-induced apoptosis and counters intracel-lular prosurvival signals from diverse proinflammatory mediatorsin human neutrophils in vitro.Our results identify RvE1 as a modulator of neutrophil apo-

ptosis, an important control point of the resolution of inflam-

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cells/mL) were cultured for 24 h with RvE1 and viability, mitochondrialtransmembrane potential (ΔΨm) [chloromethyl-X-rosamine (CMXRos) stain-ing] and apoptosis (annexin-V–FITC binding and nuclear DNA content) wereassessed. (A) RVE1 at high concentrations suppresses intrinsic apoptosis. Dataare means ± SEM (n = 4–11). *P < 0.05 vs. untreated. (B) To monitor ROSproduction, neutrophils were loaded with H2DCFDA (5 μM) and then leftuntreated (C, control) or challenged with RvE1 for 15 min. Data are means ±SEM (n = 4–7). *P < 0.05; **P < 0.01; ***P < 0.001 vs. untreated. (C) Caspase-8 activity was assessed at 4 h post-RvE1 with flow cytometry using FITC-la-beled z-Ile-Glu(Ome)-Thr-Asp(Ome)-fluoromethylketone (z-IETD-fmk) asa substrate. The effect of the Fas-activating antibody (CH-11 Ab) is shown forcomparison. (n = 4–7). *P < 0.05; **P < 0.01 vs. untreated.

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mation (2, 5) and provide several insights into the underlyingmolecular mechanisms. At concentrations ≤ 10 nM, RvE1 per sedid not affect the constitutive death program in neutrophils (17,and the present study), whereas it activated intracellular pathwaysto enhance phagocytosis-induced apoptosis and to counter pro-survival signals. However, RvE1 at 100–1,000-nM concentrationrange evoked modest increases in ROS production and caspase-8activity, but also triggered competing survival signals throughsustained ERK and Akt activation, shifting the balance in neu-trophils toward survival. These latter actions ofRvE1 resemble theactions of GM-CSF that delay neutrophil apoptosis (21).Phagocytosis of complement-opsonized targets requires Mac-1

and induces apoptosis via NADPH oxidase-derived ROS-medi-ated activation of caspase-8 (21). This occurs despite ERK ac-tivation following Mac-1 ligation (32), indicating that cross-talkbetween intracellular pro- and anti-apoptotic signals generatedin phagocytosing neutrophils places the balance on the apoptoticpathway triggered by phagocytosis. Our findings show that RvE1enhances phagocytosis of opsonized bacteria and yeast, which, inturn, leads to increased ROS generation by NADPH oxidase andactivation of caspase-8 and caspase-3, reinforcing the shift ofbalance toward apoptosis. This conclusion relies on pharmaco-logical inhibition of NADPH oxidase with DPI. Although DPIalso inhibits other flavoprotein-using enzymes, including nitricoxide (NO) synthase, NO is not essential for phagocytosis-in-duced neutrophil apoptosis (30). Of note, RvE1 in the concen-tration range of 1–10 nM was reported to enhance phagocytosis

of zymosan, live E. coli, and apoptotic neutrophils by human andmurine macrophages, leading to a macrophage phenotype switchwithout evoking apoptosis (13, 14, 17).Earlier studies showed that MPO signaling through Mac-1 (6),

SAA through FPR2/ALX (26) and CpG DNA through TLR-9(27) generate survival cues for neutrophils through concomitantactivation of the ERK and Akt pathways, leading to preventionof Mcl-1 degradation and collapse of mitochondrial function. Inhuman neutrophils, RvE1 produced modest decreases in ERKand Akt phosphorylation by MPO, SAA, or CpG DNA andevoked ROS generation and caspase-8/caspase-3 activation,consistent with enhanced apoptosis. Complete blockade of theRvE1 actions by apocynin and DPI lend additional support forRvE1 activation of the NADPH oxidase-derived ROS-caspase-8proapoptosis circuit. Of note, RvE1 also decreased MPO-stim-ulated up-regulation of Mac-1, implicating disruption of anMPO-mediated autocrine and paracrine loop for perpetuation ofthe inflammatory response (6).RvE1 can interact with the LTB4 receptor BLT1 and the

ChemR23 receptor (15, 31), both of which may mediate celltype–specific actions. In contrast to BLT1, human neutrophilsexpress low levels of ChemR23 mRNA, but the expression andfunction of the ChemR23 protein is still uncertain (14, 15). Inour experiments, ChemR23 protein expression was undetectablewith flow cytometry. RvE1 has been reported to act as a partialagonist on BLT1 and to antagonize the proinflammatory actionsof LTB4 mainly through inhibition of neutrophil trafficking (15).

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Fig. 2. RvE1 enhances phagocytosis-induced neutrophil apoptosis. (A and B) Human neutrophils (5 × 106 cells/mL) were mixed with opsonized FITC-labeled E.coli at a ratio of 1:10 for 1, 2, and 4 h. Extracellular fluorescence was quenched with 0.2% trypan blue and intracellular fluorescence was analyzed with flowcytometry (A) or fluorescence microscopy (B). Inset: representative time course of neutrophil phagocytosis of bacteria. Results are expressed as percentageincrease above vehicle plus E. coli (n = 4). (C) Neutrophils were cultured for 24 h with yeast (five yeast particles/neutrophil) with or without RvE1 (10 nM),U75302 (1 μM), LY255238 (1 μM), or DPI (20 μM), stained with acridine orange (10 μg/mL), and apoptosis was assessed by nuclear morphology (condensed orfragmented chromatin) under a fluorescence microscope. Data are means ± SEM (n = 5–6). *P < 0.05; **P < 0.01. (D) ROS generation. The effects of RvE1 areexpressed as percentage increase above vehicle plus yeast. Inset: Time course of ROS production by phagocytosing neutrophils. Data are means ± SEM (n = 5).Caspase-8 (E) and caspase-3 (F) activity was assessed at 4 h post-RvE1 with flow cytometry using FITC-labeled z-IETD-fmk and acetyl-Asp-Glu-Val-Asp-fluo-romethylketone (Ac-DEVD-fmk), respectively, as substrates. Data are means ± SEM (n = 6). *P < 0.05; **P < 0.01; ***P < 0.001.

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Our results with selective LTB4-receptor antagonists now identifyBLT1 as the predominant receptor mediating the apoptosis pro-moting action of RvE1 in vitro, indicating that resolution mech-anisms may also be activated via BLT1. Thus, RvE1 may exertdifferent proresolution actions via distinct receptors. It promotesneutrophil apoptosis through BLT1 and enhances phagocytosis ofapoptotic cells through ChemR23 (14, 33), which suggests thatconcurrent actions on both receptors may be critical for optimalresolution. It is interesting to note that clearance of apoptotic cellsvia ChemR23 also occurs at similar low- or subnanomolar RvE1concentrations (14, 17, 34) as those required to induce neutrophilapoptosis.Extending observations in mouse models of aspiration pneu-

monia and asthma (18–20), we also found that RvE1 facilitatesresolution of inflammation in clinically relevant mouse models ofALI/ARDS; i.e., intratracheal instillation of E. coli or carrageenan

plus MPO and E. coli septicemia–associated acute lung injury.Although the benefits of a diet enriched with EPA have beenrecognized in reducing disease severity and mortality in patientswith ALI or acute respiratory distress syndrome (35, 36) parallel

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Fig. 3. RvE1 attenuates MPO suppression of neutrophil apoptosis. (A) Hu-man neutrophils (5 × 106 cells/mL) were cultured for 20 min with RvE1 thenwith MPO (160 nM) for 24 h. Viability, mitochondrial transmembrane po-tential (ΔΨm) (CMXRos staining), and apoptosis (annexin-V–FITC binding andnuclear DNA content) were then assessed. Data are means ± SEM (n = 4–7).*P < 0.05; **P < 0.01; ***P < 0.001 vs. untreated (C, control). #P < 0.01 vs.MPO-treated. (B) Impact on MAP kinases and Mcl-1. Neutrophils were lysedafter culture with MPO (160 nM) ± RvE1 (10 nM) for 30 min (MAP kinases) or1 h (for Mcl-1). Proteins were subjected to immunoblotting with antibodiesto phosphorylated kinases, Mcl-1, or actin. Numerical values indicate relativedensity of bands normalized with density of actin bands. Results are repre-sentative of three separate experiments.

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with reduced neutrophil accumulation in the lung (37), the cur-rent results raise the possibility that EPA-derived RvE1 mediatedsome of these clinical effects. MPO, SAA, and CpG DNA havebeen implicated in mediating E. coli pneumonia and E. coli sep-ticemia–associated lung injury (23–25), whereas carrageenanevokes self-resolving pulmonary inflammation that can be pro-longed by coadministration of MPO (6). Our results indicate thatenhanced resolution of inflammation by RvE1 was intimatelylinked to caspase-dependent apoptosis of neutrophils within theairways. Thus, RvE1 promoted apoptosis in large proportions ofBAL fluid neutrophils. Furthermore, the pan-caspase inhibitorzVAD-fmk prevented RvE1-induced dramatic drop in the num-ber of neutrophils in the airways and pulmonary tissues, indicatingthat neutrophil apoptosis is required for their timely clearance,

and even aggravated lung injury likely due to persisting presenceof neutrophils.PretreatmentwithRvE1has been reported to reduce neutrophil

recruitment in response to inflammatory stimuli (reviewed in 31).We now show that a bolus injection of RvE1 administered at nearpeak of inflammation was efficient to affect neutrophils withinthe lung, indicating its therapeutic potential. Thus, RvE1 attenu-ation of neutrophil trafficking combined with enhanced apoptosisand clearance contribute to its proresolution properties. Fur-thermore, RvE1 also reduced vascular permeability and release ofthe proinflammatory cytokine IL-6, consistent with an anti-inflam-matory role. In our ALImodels, we observed increased number ofmonocytes/macrophages in the airways in response toRvE1,whichis consistent with facilitation of tissue repair (2, 38, 39) and theoriginal properties defining RvE1 actions (12, 40). The high per-centage of macrophages with apoptotic bodies indicates enhancedclearance by RvE1 of apoptotic neutrophils and other cells.Phagocytosis of apoptotic cells induces the release of mediatorswith proresolution and repair properties, such as IL-10 andTGF-β(2, 38), which contributes to the resolution.Our in vitro and in vivo work shows that RvE1 shifts the balance

between competing pro- and anti-apoptotic signals toward apo-ptotic pathways, thereby affecting the fate of emigrated neu-trophils. RvE1 promotion of neutrophil apoptosis representsa clinically relevant mechanism for facilitating removal of emi-grated neutrophils as we demonstrated here in three mousemodels of ALI. Neutrophils will likely encounter RvE1 duringinflammation when they have already received prosurvival cuesfrom the inflammatory microenvironment and being engaged inphagocytosing opsonized pathogens. Thus, RvE1 joins the cyclin-dependent kinase inhibitor drugs (7, 8), 15-epi-LXA4 (9) andTNF-related apoptosis-inducing ligand (41) as neutrophil apo-ptosis-inducing agent, but exerts its actions through different andunique molecular mechanisms.Taken together, these data provide evidence for a mechanism,

facilitation of neutrophil apoptosis, by which eicosapentaenoicacid-derived RvE1 could promote resolution of ALI/ARDS. Wefound that RvE1 via the BLT1 receptor promoted phagocytosis-induced neutrophil apoptosis and attenuated anti-apoptosis sig-nals fromMPO, SAA, and CpGDNA in vitro. RvE1 administeredat a clinically relevant time point, near the peak of inflammationconsistently enhanced resolution of lung inflammation in threemurine models of ALI by enhancing apoptosis in emigrated neu-trophils and facilitating their removal by macrophages. Our find-ings reinforce the concept of therapeutic targeting of neutrophilapoptosis for enhancing the resolution of ALI/ARDS and otherinflammatory pathologies.

Materials and MethodsNeutrophil Culture, Assessment of Apoptosis, and Phagocytosis. Human neu-trophils (purity >96%, apoptotic <3%), resuspended in HBSS containing 10%(vol/vol) autologous serum, were cultured with RvE1 (0.4–1,000 nM; ResolvyxPharmaceuticals) and then challengedwithMPO (160 nM), SAA (10 μg/mL), orE. coli DNA (CpG DNA, 1.6 μg/mL) with or without apocynin (100 μM) ordiphenyleneiodonium chloride (20 μM). Apoptosis was assessed by annexin-Vstaining, nuclear morphology, DNA cleavage, and intracellular caspase-3 andcaspase-8 activity (6, 26). For quantitative analysis of phagocytosis, neutrophilswere mixed with opsonized FITC-labeled E. coli at a ratio of 1:10 and in-tracellular fluorescence was analyzed (21). To assess apoptosis, neutrophilswere cultured for 24 h with Saccharomyces cerevisae (five yeast particles/neutrophil) with or without RvE1 (10 nM), the LTB4 receptor BLT1 antagonistU75302 (1 μM), the BLT2 receptor antagonist LY255238 (1 μM), or DPI (20 μM),and the percentage of neutrophils with apoptotic nuclei was determined.

Murine Acute Lung Injury. Female BALB/c mice (aged 8–12 wk; Charles RiverLaboratories) were injected intraperitoneally 2 × 108 −109 live E. coli (9, 23)or intratracheally with 107 live E. coli or 0.1 mL of 0.25% λ-carrageenan plus10 μL of 16-μM MPO (6). At the peak of inflammation, mice were treatedwith RvE1 (25 μg/kg, i.p.) or the pan-caspase inhibitor z-Val-Ala-DL-Asp-flu-oromethylketone (zVAD-fmk; 10 μg/kg, three times at 4-h intervals). At theindicated times, the lungs were lavaged or processed for histology withoutlavage. BAL fluid leukocyte count, protein, IL-6 levels, and neutrophil

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Fig. 5. RvE1 enhances resolution of E. coli–evoked pneumonia. Six hoursafter intratracheal instillation of 107 live E. coli, mice were treated withvehicle or RvE1 (25 μg/kg, i.p.) and/or the pan-caspase inhibitor zVAD-fmk(10 μg/kg, i.p. three times at 4-h intervals). Mice were killed 24 h later andlung myeloproxidase content (A), BAL fluid protein (B), lung dry-to-wetweight ratio (C), and BAL fluid IL-6 level (D) were determined. Data aremeans ± SEM (n = 6–8 mice per group). *P < 0.05, **P < 0.01; ***P < 0.001.(E) Lung tissue sections from naive mice (control), mice with inflammationevoked by E. coli, or mice treated with vehicle, RvE1, and/or zVAD-fmk for 24 h.Hematoxylin and eosin stain (H&E); scale bars: 100 μm.

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apoptosis were determined (6). Full details and other methods are describedin SI Materials and Methods.

Statistical Analysis. Values represent mean ± SEM. Statistical comparisonswere made by ANOVA using ranks followed by Dunn’s multiple contrasthypothesis tests, the Wilcoxon signed rank test or by the Mann-Whitney u

test. P <0.05 were considered statistically significant. Kaplan-Meyer survivalcurves were compared using the log-rank test.

ACKNOWLEDGMENTS. We thank Dr. Charles N. Serhan for helpful discussionsandcritical readingof themanuscript. Thisworkwas supportedbyGrantMOP-97742 (to J.G.F.) from the Canadian Institutes of Health Research.

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Fig. 6. RvE1 attenuates lung inflammation andprolongs survival of mice with E. coli peritonitis. Onehour after i.p. injection of 2 × 108 live E. coli, micewere treated with vehicle or RvE1 (25 μg/kg, i.p.).Mice were killed at 6-h post–E. coli and BAL fluidneutrophil counts (A), the percentage of annexin-V–positive (apoptotic) neutrophils (B), BAL fluid cellcytoplasmic histone-associated DNA fragments (C),and the percentage of macrophages containing apo-ptotic bodies (D) were determined. Data are means ±SEM (n = 6 mice per group). *P < 0.05, **P < 0.01. (E)Lung tissue sections from naive mice (control) andmice challenged with E. coli and treated with RvE1 orvehicle. H&E stain; scale bars: 100 μm. (F) Kaplan-Meier survival plots for mice challenged intraperi-toneally with 109 live E. coli and treated with RvE1or vehicle (n = 10 per group). P = 0.0315 by the log-rank test.

14988 | www.pnas.org/cgi/doi/10.1073/pnas.1206641109 El Kebir et al.

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